Identification and antigenicity of the Babesia caballi spherical body protein 4 (SBP4)

Authors: MAHMOUD, M - Egypt National Research Center; KANDIL, O - Egypt National Research Center; ABU EL-EZZ, NADIA - Egypt National Research Center; HENDAWY, SEHAM - Egypt National Research Center; ELSAWY, BASSMA - Egypt National Research Center; KNOWLES, DONALD - Washington State University; BASTOS, REGINALDO - Washington State University; Kappmeyer, Lowell; LAUGHERY, JACOB - Washington State University; ALZAN, HEBBA - Washington State University; Suarez, Carlos

Submitted to: Parasites & Vectors
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/15/2020
Publication Date: 7/22/2020
Citation: Mahmoud, M.S., Kandil, O.M., Abu El-Ezz, N.T., Hendawy, S.H.M., Elsawy, B.S.M., Knowles, D.P., Bastos, R.G., Kappmeyer, L.S., Laughery, J.M., Alzan, H.F., Suarez, C.E. 2020. Identification and antigenicity of the Babesia caballi spherical body protein 4 (SBP4). Parasites & Vectors. 13. Article 369. https://doi.org/10.1186/s13071-020-04241-9.
DOI: https://doi.org/10.1186/s13071-020-04241-9

Interpretive Summary: The tick-borne Babesia caballi is the etiological agent of equine babesiosis, an economically important disease of equids in most tropical and subtropical areas of the world. Discovering candidate antigens for improved diagnostic tools and vaccines remains a need for improved control of equine babesiosis. The widely conserved spherical body protein 4 (SBP4), in Babesia and Theileria, was previously identified as a promising candidate for the diagnosis of bovine babesiosis. This study describes B. caballi sbp4 (Bcsbp4) gene, analyzed its pattern of expression, and verified the antigenicity of B. caballi SBP4 (BcSBP4). Sera from 8 equids that were infected with B. caballi, but not from a set of 10 non-infected equids, gave a positive signal in rBcSBP4 based diagnostic serological test. The findings indicate that a well conserved sbp4 gene is expressed in B. caballi blood stages. The SBP4 is a potential candidate for developing an improved serological test that could detect B. caballi infection in tropical and subtropical countries worldwide.

Technical Abstract: Background: The tick-borne intra-erythrocytic apicomplexan Babesia caballi is the etiological agent of equine babesiosis, an economically important disease of equids in most tropical and subtropical areas of the world. Discovering candidate antigens for improved diagnostic tools and vaccines remains a need for improved control of equine babesiosis. The widely conserved spherical body protein 4 (SBP4), in Babesia and Theileria, was previously identified as a promising candidate for the diagnosis of bovine babesiosis. This study describes B. caballi sbp4 (Bcsbp4) gene, analyzed its pattern of expression, and verified the antigenicity of B. caballi SBP4 (BcSBP4). Methods: BLAST searches of an uncurated B. caballi assembly genome using the B. bovis SBP4 as a query followed by amplification by PCR and sequencing of BcSBP4. We performed bioinformatics analysis, immunoblots, immunofluorescence and an in vitro neutralization test using anti SBP4 peptide antibodies. Antigenicity of recombinant BcSBP4 (rBcSBP4) was tested with sera from field animals (n=18) using an indirect ELISA (iELISA). Results: B. caballi genome searching using B. bovis SBP4 as a query resulted in identification of Bcsbp4. The Bcsbp4 gene encodes for a protein of 27 kDa, which is fully conserved among B. caballi isolates from US and Egypt. Bioinformatics analysis indicates that BcSBP4 contains a signal peptide and lacks additional TM domains. Expression of BcSBP4 in blood stages was confirmed by Western blot and immunofluorescence (IFA) using antibodies against synthetic peptides representing putative B-cell epitopes of BcSBP4 predicted by in silico analysis. In vitro neutralization test showed a marginal, but statistically significant inhibitory effect on the infectivity of B. caballi merozoites. Sera from 8 equids that were judged to be infected with B. caballi, but not from a set of 10 non-infected equids, gave a positive signal in rBcSBP4 based iELISA.